Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of art as known to those skilled therein as of the date of the invention described and claimed herein.
Superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is the enzyme that catalyzes the removal of superoxide radicals, which are generated in a variety of biological oxidations (1). It provides a defense against oxygen toxicity and damage that may be caused to cells by carcinogenic hydrocarbons (1). The human Cu-Zn superoxide dismutase (SOD-1) is a dimeric protein composed of apparently identical noncovalently linked subunits, each with a molecular weight of 16,000-19,000 (2,3). The locus for human cytoplasmic superoxide dismutase (SOD-1) was assigned to chromosome 21 (4).
About 1 in 600 newborn babies carries an extra chromosome 21, a condition technically known as trisomy 21 or Down syndrome (5,6). This chromosome imbalance is a known cause of spontaneous abortion and mental retardation (5). In most cases, the patients with Down syndrome have karyotypes with 47 chromosomes (46 plus one additional 21). However, cases of Down syndrome in which only a portion of chromosome 21 is present in triplicate have enabled the localization of the "responsible" region to segment 21q22, the distal portion of the long arm (7-11). Although trisomy 21 was identified as a human genetic disease over 20 years ago (5), little is known about the mechanisms by which the extra chromosome or the extra chromosomal segment 21q22 results in reduced viability and abnormalities of morphogenesis and mental function. It is generally assumed that the extra chromosome or chromosomal segment codes for normal products and that the abnormalities found in Down syndrome are produced by an imbalance due to changes in gene dosage (12). Namely, the presence of additional genetic material in the cell will result in the production of commensurately increased amounts of the gene products coded by the extra chromosomal segment. Indeed, Down syndrome patients show an increase of about 50% in SOD-1 activity (13-15) due to a higher level of SOD-1 protein (16). However, it is not known whether this gene dosage phenomenon is a result of quantitative changes in the amount of SOD-1 mRNA.
In the past, most of the reports on Down syndrome involved family karyotyping and clinical studies of the effects of the disease on patients. It is only recently that recombinant DNA techniques have enabled one to approach the molecular biology of the chromosomal region involved and try to gain insight into the mechanism by which abnormal karyotypes result in abnormal phenotype.
Superoxide dismutase is also of interest because of its pharmacological properties. Bovine-derived superoxide dismutase (orgotein) has been recognized to possess anti-inflammatory properties and is currently marketed in parts of Europe as a human pharmaceutical. It is also sold in the United States as a veterinary product, particularly for use with horses. However, supplies of orgotein are limited. Prior techniques involving recovery from bovine or other animal cells have serious limitations and the orgotein so obtained may produce allergic reactions in humans because of its non-human origin.
To meet these various needs, efforts were undertaken to identify the gene encoding human cytoplasmic superoxide dismutase and to prepare a cDNA molecule containing such a gene. These efforts resulted in the present invention which is described more fully hereinafter. A description of certain aspects of the invention was published in Proc. Natl. Acad. Sci., USA, vol. 79, pp. 2808-2811, May 1982 (30).